Clinical listonellosis in meagre ( Argyrosomus regius ) from recirculated aquaculture system in Turkey

Vibriosis caused by Listonella anguillarum was reported in several fish species from both fresh and saltwater conditions. This pathogen causes disease in rainbow trout, sea bass, and sea bream in Turkey, however, it has not been reported from meagre (Argyrosomus regius) before. Great loss of meagre was observed in the Recirculated Aquaculture System at the Faculty of Fisheries of Izmir Katip Celebi University, which had been transferred from a commercial hatchery for a nutrition experiment. Clinical signs of vibriosis were observed in infected fish, i.e. haemorrhage in the anal area and pectoral fins, mostly as tail ulcers. Petechial haemorrhages in the muscle, liver, peritoneal membranes and pyloric caeca were determined by necropsy. A Gramnegative, rod-shaped, motile bacterium was isolated, showing a positive reaction to oxidase, catalase and gelatin tests, and being sensitive to O/129. Biochemical identification tests and PCR amplifications identified the bacterium as Listonella anguillarum. In slide agglutination test with anti L. anguillarum O1 (ATCC43305) serum, all isolates were positive. The isolated bacteria was resistant to oxytetracycline, sensitive to enrofloxacin, flumequine, phosphomycin, furozulidone, kanamycin and oxolinic acid. In this study, the isolated bacteria from meagre were determined as Listonella anguillarum O1 with biochemical, moleculer identification and agglutination tests. Listonella anguillarum, fish disease, isolation Vibriosis that has been found in more than 50 fresh and saltwater fishes is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. The first known vibriosis outbreak was reported in 1893 in eels as Bacterium anguillarum (Canestrini 1907). In 1909, Bergman proposed the name of Vibrio anguillarum but in 1985, Mac Donell and Colwell reclassified the pathogen into the new genus, Listonella. Currently, both nomenclatures are present in the literature (Hickey and Lee 2017). Listonellosis, also known as salt-water furunculosis (Rucker 1963), boil disease (Kubota and Takakuwa 1963) and ulcer disease (Bagge and Bagge 1956), is caused by a Gram-negative, polar flagellated, comma-shaped rod bacterium Listonella (Vibrio) anguillarum from the Vibrionaceae family (Actis et al. 1999). It grows on rich media containing 1.5–2.0% sodium chloride (NaCl) between 25–30°C temperatures and forms cream-coloured, round-shaped colonies (Frans et al. 2011). Chemical stress such as water quality, diet composition and pollution, biological stress like population density and the presence of other macroor micro-organisms, and physical stress due to temperature and overcrowding are important factors that cause outbreaks (Thune et al. 1993; Frans et al. 2011). In addition, the virulence of the L. anguillarum strains influences the onset of disease outbreaks (Actis et al. 1999). It has been postulated that in acute epizootics, infected fish dies rapidly without showing any clinical signs (Actis et al. 1999; Toranzo et al. 2005; Austin and Austin 2007; Frans et al. 2011). In Turkey, L. anguillarum was reported in cultured sea bass (Çağırgan 1993) and rainbow trout (Oncorhynchus mykiss) (Tanrıkul 2007). Currently, additional isolates were ACTA VET. BRNO 2018, 87: 269-275; https://doi.org/10.2754/avb201887030269 Address for correspondence: Ezgi Dinçtürk Izmir Katip Celebi University Faculty of Fisheries Balatçık Mahallesi Havaalanı Şosesi No:33/2 Balatçık 35620 Çiğli İzmir, Turkey Phone: +90 5396832489 E-mail: ezgi.dincturk@ikc.edu.tr http://actavet.vfu.cz/ described from different fish farms and species as a result of the expanding aquaculture industry. Meagre (Argyrosomus regius) farming started in Turkey in 2007 and now it is one of the alternative species that have increased production numbers because of its favourable biological characteristics such as being resistant to environmental conditions, having good feed conversion, fast growth, and high flesh quality. The objective of this study was to determine and to identify the biochemical characteristics of Listonella anguillarium serogroup O1 from meagre. This pathogen has been known for some time but has not been reported previously in Turkey. Materials and Methods Fish The outbreak was observed in the Recirculated Aquaculture System at Faculty of Fisheries of the Izmir Katip Celebi University. The fish were supplied to the university from a commercial hatchery for a nutrition experiment. The water source of the system was brackish (3.9‰) water and the temperature was 16 ±2 °C when high mortality was observed. Bacterial strains Isolation and identification of bacteria A total of 25 moribund fish of 20 g were examined by bacteriological investigations. The isolates from the kidneys of diseased fish were streaked on tryptone soy agar (TSA, Oxoid, UK) including 1.5% NaCl and incubated at 21 °C for 48 h. After primer isolation, the representative colonies were passaged onto thiosulphatecitrate-bile salts-sucrose agar (TCBS, Oxoid, UK). The purified colonies were cultivated on TSA including 1.5% NaCl for biochemical tests. Gram staining and oxidase test were carried out according to standard procedures. The motility of the bacteria was detected by the hanging-drop technique. Oxidative and fermentative degradation of glucose and gas production were tested on O/F Medium (Difco, USA). Resistance to the 2,4-diamino-6,7diisopropylpteridine O/129 vibriostatic agent was tested with discs (Oxoid, UK) on TSA (Oxoid, UK) including 1.5% NaCl. Biochemical tests for further information were carried out with API 20E (BioMerieux S. A., France) (Tanrikul et al. 2005) which was carried out at 21°C for 48 h. Sterile 1.5% saline was used for inoculation of the bacterium to API 20E strips. Preparation of antisera and slide agglutination test Slide agglutination test was performed according to Toranzo et al. (1983). Rabbit serum was used against all isolated L. anguillarım O1 strains (ATCC 43305). Bacterial growth occurred on brain heart infusion agar (BHIA, Oxoid, UK) plates at 25 °C for 24–28 h. Bacterial strains were resuspended in PBS (phosphate buffered saline solution) and reached the concentration of McFarland Standard No. 3 for use as antigens in the slide agglutination tests. Molecular identification of strains Molecular identification of the bacteria was conducted. The 16S rRNA gene sequence was amplified by polymerase chain reaction (PCR) in order to confirm that the bacteria were Listonella anguillarum. The pathogen was obtained from the samples that were isolated from infected meagre during the outbreak. EurX GeneMATRIX Tissue Bacteria Isolation Kit (EURx Ltd., Poland) was used for DNA isolation. Then with use of Nanodrop 2000 (Thermo Scientific, USA), density and quality of the isolates were determined. The 27F and 1492R primers were used for PCR amplifications. Band screening of the PCR products was observed in the gel electrophoresis. Amplified products of template DNA were sent to the Stab Vida direct sequencing service (Spain) with the ABI 3730 XL DNA Analyzer for sequence determination. Then the sequences were checked against the BLASTN 2.6.1. database. Antimicrobial sensitivity test Antibiotic susceptibility of the pathogen was tested with 8 antibiotics on Mueller-Hilton agar (MHA, Merck, Germany) using the Kirby-Bauer disc diffusion technique (Stokes et al. 1993). A loopful of 24 h culture was placed in the center of a Petri dish with media and spread with a dry swab. Test discs of enrofloxacin, florfenicol, flumequine, phosphomycin, furozulidone, kanamycin, oxolinic acid, oxytetracycline and sulphamethoxazole/ trimethoprim (Oxoid, UK) were placed on the dishes and incubated at 21 °C for 24 h. The disk diffusion zone diameters were measured (mm) and compared with the interpretive criterias of National Commite for Clinical Laboratory Standarts (NCCLS, 1993, 1994).

Vibriosis that has been found in more than 50 fresh and saltwater fishes is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio.The first known vibriosis outbreak was reported in 1893 in eels as Bacterium anguillarum (Canestrini 1907).In 1909, Bergman proposed the name of Vibrio anguillarum but in 1985, Mac Donell and Colwell reclassified the pathogen into the new genus, Listonella.Currently, both nomenclatures are present in the literature (Hickey and Lee 2017).
Listonellosis, also known as salt-water furunculosis (Rucker 1963), boil disease (Kubota and Takakuwa 1963) and ulcer disease (Bagge and Bagge 1956), is caused by a Gram-negative, polar flagellated, comma-shaped rod bacterium Listonella (Vibrio) anguillarum from the Vibrionaceae family (Actis et al. 1999).It grows on rich media containing 1.5-2.0%sodium chloride (NaCl) between 25-30°C temperatures and forms cream-coloured, round-shaped colonies (Frans et al. 2011).Chemical stress such as water quality, diet composition and pollution, biological stress like population density and the presence of other macro-or micro-organisms, and physical stress due to temperature and overcrowding are important factors that cause outbreaks (Thune et al. 1993;Frans et al. 2011).In addition, the virulence of the L. anguillarum strains influences the onset of disease outbreaks (Actis et al. 1999).It has been postulated that in acute epizootics, infected fish dies rapidly without showing any clinical signs (Actis et al. 1999;Toranzo et al. 2005;Austin and Austin 2007;Frans et al. 2011).
In Turkey, L. anguillarum was reported in cultured sea bass (Çağırgan 1993) and rainbow trout (Oncorhynchus mykiss) (Tanrıkul 2007).Currently, additional isolates were described from different fish farms and species as a result of the expanding aquaculture industry.Meagre (Argyrosomus regius) farming started in Turkey in 2007 and now it is one of the alternative species that have increased production numbers because of its favourable biological characteristics such as being resistant to environmental conditions, having good feed conversion, fast growth, and high flesh quality.
The objective of this study was to determine and to identify the biochemical characteristics of Listonella anguillarium serogroup O1 from meagre.This pathogen has been known for some time but has not been reported previously in Turkey.

Fish
The outbreak was observed in the Recirculated Aquaculture System at Faculty of Fisheries of the Izmir Katip Celebi University.The fish were supplied to the university from a commercial hatchery for a nutrition experiment.The water source of the system was brackish (3.9‰) water and the temperature was 16 ±2 °C when high mortality was observed.

Bacterial strains Isolation and identification of bacteria
A total of 25 moribund fish of 20 g were examined by bacteriological investigations.The isolates from the kidneys of diseased fish were streaked on tryptone soy agar (TSA, Oxoid, UK) including 1.5% NaCl and incubated at 21 °C for 48 h.After primer isolation, the representative colonies were passaged onto thiosulphatecitrate-bile salts-sucrose agar (TCBS, Oxoid, UK).The purified colonies were cultivated on TSA including 1.5% NaCl for biochemical tests.Gram staining and oxidase test were carried out according to standard procedures.The motility of the bacteria was detected by the hanging-drop technique.Oxidative and fermentative degradation of glucose and gas production were tested on O/F Medium (Difco, USA).Resistance to the 2,4-diamino-6,7diisopropylpteridine O/129 vibriostatic agent was tested with discs (Oxoid, UK) on TSA (Oxoid, UK) including 1.5% NaCl.Biochemical tests for further information were carried out with API 20E (BioMerieux S. A., France) (Tanrikul et al. 2005) which was carried out at 21°C for 48 h.Sterile 1.5% saline was used for inoculation of the bacterium to API 20E strips.

Preparation of antisera and slide agglutination test
Slide agglutination test was performed according to Toranzo et al. (1983).Rabbit serum was used against all isolated L. anguillarım O1 strains (ATCC 43305).Bacterial growth occurred on brain heart infusion agar (BHIA, Oxoid, UK) plates at 25 °C for 24-28 h.Bacterial strains were resuspended in PBS (phosphate buffered saline solution) and reached the concentration of McFarland Standard No. 3 for use as antigens in the slide agglutination tests.

Molecular identification of strains
Molecular identification of the bacteria was conducted.The 16S rRNA gene sequence was amplified by polymerase chain reaction (PCR) in order to confirm that the bacteria were Listonella anguillarum.The pathogen was obtained from the samples that were isolated from infected meagre during the outbreak.EurX GeneMATRIX Tissue Bacteria Isolation Kit (EURx Ltd., Poland) was used for DNA isolation.Then with use of Nanodrop 2000 (Thermo Scientific, USA), density and quality of the isolates were determined.The 27F and 1492R primers were used for PCR amplifications.Band screening of the PCR products was observed in the gel electrophoresis.Amplified products of template DNA were sent to the Stab Vida direct sequencing service (Spain) with the ABI 3730 XL DNA Analyzer for sequence determination.Then the sequences were checked against the BLASTN 2.6.1.database.

Antimicrobial sensitivity test
Antibiotic susceptibility of the pathogen was tested with 8 antibiotics on Mueller-Hilton agar (MHA, Merck, Germany) using the Kirby-Bauer disc diffusion technique (Stokes et al. 1993).A loopful of 24 h culture was placed in the center of a Petri dish with media and spread with a dry swab.Test discs of enrofloxacin, florfenicol, flumequine, phosphomycin, furozulidone, kanamycin, oxolinic acid, oxytetracycline and sulphamethoxazole/ trimethoprim (Oxoid, UK) were placed on the dishes and incubated at 21 °C for 24 h.The disk diffusion zone diameters were measured (mm) and compared with the interpretive criterias of National Commite for Clinical Laboratory Standarts (NCCLS, 1993(NCCLS, , 1994)).

Results
The affected meagre exhibited erratic swimming, dark discolouration, haemorrhage in the anal area and pectoral fins, and tail ulcers as clinical signs.High mortality was observed and calculated as 30% during the outbreak.Necropsy showed petechial haemorrhage in the muscle, liver, peritoneal membranes and pyloric caeca from the internal sides of the fish (Plate VIII, Fig. 1).
The morphologic and biochemical properties of isolated L. anguillarum O1 are presented in Table 1.The isolates were found to be Gramnegative, motile, oxidase and catalase positive.
The PCR amplification of the L. anguillarum gene sequence was registered in the BLASTN 2.6.1 database.It resulted in 100% nucleotide identity between the current isolate and Listonella anguillarum (accession number CP023208.1).
The antibiotic susceptibility results of isolated L. anguillarum O1 strains are presented in Table 2.The pathogen showed resistance to oxytetracycline and it was susceptible to enrofloxacin, flumequine, phosphomycin, furozulidone, kanamycin and oxolinic acid.The intermediate intensity of antibiotics were detected in florfenicol and sulphamethoxazole/trimethoprim test groups.
Infected fish were treated with enrofloxacin added to dry pellet feed (50 mg•kg -1 fish per day) for 7 consecutive days with 1.3% feed ratio.
Meagre was indicated as fast-growing, fairly fecund and longlived but it has been reported that the biology, ecology and fisheries of this species are poorly documented, especially in European waters (Quéméner 2002;Prista 2013).In recent years it has been considered as a potential candidate for culture in Turkey.Diseases caused by L. anguillarum were detected in both hatcheries and cages with increasing production rates.Juveniles were destroyed and adult individuals were treated with antibiotics without determining the pathogen to fight the disease.The disease has been known in hatchery and cage culture of meagre in Turkey but has not been reported before.Therefore, this study is the first declaration of Listonella anguillarum O1 from Argyrosomus regius in Turkey.
Phenotypic characteristics like Gram staining, motility, oxidase and catalase reactions of L. anguillarum strains were detected in accordance with Austin and Austin (2007).The API 20E rapid identification systems have been used for L. anguillarum strains (Tanrıkul et al. 2005;Tanrıkul 2007).According to the API 20E results the isolate was identified as L. anguillarum and no biochemical differences were detected on the isolated L.anguillarum strains.These findings are similar to Tanrikul et al (2005), Demircan and Candan (2006), Korun (2006) and Avsever (2014) for different marine species, mostly sea bass (Dicentrarchus labrax) as well as isolations from rainbow trout (Tanrikul 2007;Tanrikul and Gultepe 2011).Some indicators such as ADH, indole, inositol, sorbitol and arabinose vary among different studies (Table 3), but biochemical results are not the essential criteria to identify the bacteria.The results of biochemical studies showed that biochemical properties in different species do not vary with the salinity of water (Table 3).
The sensitivity of L. anguillarum strains to chemotherapeutics has been decreasing so far (Takahashi et al. 1976).In antimicrobial susceptibility tests, resistance to oxytetracycline was detected in the bacteria.Tanrikul (2007) reported that some L. anguillarum strains isolated from rainbow trout showed resistance to enrofloxacin and oxytetracycline.On the contrary, the strains isolated from aquarium catfish were determined sensitive to enrofloxacin and oxytetracycline, but resistant to penicillin (Rad and Shahsavani 2010).These results showed that antimicrobial susceptibility may vary depending on the strain.In addition, L. anguillarum which is a fish pathogen is not included in the EUCAST list, so these results could not be compared with their criteria but the zone diameters were resulted according to the NCCLS criteria..In conclusion, there is not sufficient information about meagre infected with L.anguillarum in literature.It was not reported in Turkey before and limited information is reported from different countries.Haenen et al. (2014) mentioned this pathogen in different species including meagre in their review but this is the first scientific research about the microbiology and identification of this pathogen from meagre.There is limited information regarding diseases in this fish species and the findings of our study may contribute to meagre health management.

Table 1 .
The morphologic and biochemical properties of isolated Listonella anguillarum

Table 3 .
The morphologic and biochemical test results of Listonella anguillarum from different studies.